3 way Ligation Issues... - (Aug/29/2008 )

dephosphorylation is like fire. Too much enzyme or too long and you will ruin your DNA. It can be as much help as is hindrance. Rule of the thumb 1 pmol of DNA is dephosphorylated by 1 Unit CIP in 60minutes, at 37 Celsius, in a volume of 50ul using buffer 3.

On principle I dephosphorylate all my vectors when ligating. However many people avoid this step when working on double digested vectors as dephosphorylation is unnecessary, to save time and for the above reason. I am not sure what to recommend. It can help, but it could easily become another problem. I guess you could try both, ligation with dephos vector and non dephos vector (assuming your DNA holds up).

The only thing I can think off right now is to redo the digestion. Try cutting down the volume of enzyme a little, use about 1.5ul, and increase the digestion time to about 2.5 hours. Give it more if you can. You can do an overnight digest.

Do you have any problems with the insert amplification? Was production of the insert good? (gel would help). How much insert DNA you have? And at what concentration.

I also assume that the SacI site and AscI site on the vector have a separation of at least 6bp (since it was not mentioned how far apart these are). Is this assumption correct? Or are the SacI and AscI sites closer together?

Also, when you do the ligation, make sure you run some of the ligation mix out onto the gel after the reaction is completed. Confirm that the fragments are ligating

-perneseblue-

QUOTE (perneseblue @ Sep 4 2008, 02:42 PM)

dephosphorylation is like fire. Too much enzyme or too long and you will ruin your DNA. It can be as much help as is hindrance. Rule of the thumb 1 pmol of DNA is dephosphorylated by 1 Unit CIP in 60minutes, at 37 Celsius, in a volume of 50ul using buffer 3.

On principle I dephosphorylate all my vectors when ligating. However many people avoid this step when working on double digested vectors as dephosphorylation is unnecessary, to save time and for the above reason. I am not sure what to recommend. It can help, but it could easily become another problem. I guess you could try both, ligation with dephos vector and non dephos vector (assuming your DNA holds up).

The only thing I can think off right now is to redo the digestion. Try cutting down the volume of enzyme a little, use about 1.5ul, and increase the digestion time to about 2.5 hours. Give it more if you can. You can do an overnight digest.

Do you have any problems with the insert amplification? Was production of the insert good? (gel would help). How much insert DNA you have? And at what concentration.

I also assume that the SacI site and AscI site on the vector have a separation of at least 6bp (since it was not mentioned how far apart these are). Is this assumption correct? Or are the SacI and AscI sites closer together?

Also, when you do the ligation, make sure you run some of the ligation mix out onto the gel after the reaction is completed. Confirm that the fragments are ligating

Thanks for the info and suggestions!

What started off as a 3-way ligation is now a one-way ligation (fingers crossed). Somehow I ligated one of my fragments into a vector without the other and it magically has a complete BglII site (I then sequenced the plasmid and found out that the vector I used was mislabeled so I'm attempting to religate the fragment into the correct vector by cutting with SacI/BglII and then cutting with AscI and BglII to insert the other fragment). It all seems very simple but for some reason I'm just getting religated vector.

Initial PCR amplification appeared to be fine (I've only got a printout of the gel as the old machine crashed... I'll never forget to back up again but at least I've got hard copies of everything). I decided to re-digest my fragments for a longer period and run a gel after each digestion to make sure it's completely digested before proceeding. I won't be able to tell if the vector is cut as I'm only losing 20bp but I can tell with the fragment inserted in the incorrect plasmid. Hopefully, this works out...